Photopolymerized sol-gel column and associated methods

ABSTRACT

A separation column and a method of making the separation column are provided. The separation column includes a separation channel and fritless separation medium in the channel. The separation medium includes a porous matrix, and the porous matrix includes a metal organic polymer, such as a photopolymer. The separation medium can be used to separate a sample of analytes.

BACKGROUND OF THE INVENTION

[0001] The invention relates in general to a separation column, and, inparticular, to a photopolymerized sol-gel column.

[0002] Capillary electrochromatography (CEC) has been regarded as a verypromising analytical separation technique that combines the efficiencyof capillary zone electrophoresis (CZE) with the selectivity of liquidchromatography. Although CEC has been applied in many different areas,packed-column preparation and low-detection sensitivity remainchallenges of this technique. Capillary columns containing small silicapackings have been the mainstay of CEC. One disadvantage of packedcolumns is the fabrication of porous frits of controlled pore sizes,lengths, and high mechanical stabilities. In response to fritfabrication problems, surface-functionalized open-tubular capillarycolumns and monolithic capillaries are being developed as variants ofpacked capillary columns. Monolithic capillary columns have receivedmuch attention because of the advantages offered in the control ofpermeability and surface charge.

[0003] A major challenge in CEC techniques is the detection of samplescontaining analytes at low concentration. The lack of sensitivity at lowconcentration stems from the small sample volume and the short opticalpath length for on-line detection. Dedicated sample preparation schemesthat enrich the target analytes before sample injection are oftennecessary in order to obtain the necessary sensitivity for manyreal-world analyses. Schemes such as solvent-solvent extraction andsolid-phase extraction are often very tedious and time-consuming.

[0004] An alternative to these schemes is on-line preconcentration. Ingas chromatography, this goal is met by passing a gas stream through acold column that is subsequently heated. In high-performance liquidchromatography (HPLC), this process is usually done by gradient HPLC inwhich the analytes are retained on the column much more strongly for thefirst solvent than for succeeding ones. On-line preconcentration hasalso enjoyed some success in electrophoretic separations. For example,in capillary electrophoresis (CE), these include isotachophoresis,sample stacking, sweeping, and the use of a dynamic pH junction. In CZE,F. E. P. Mikkers, F. M. Everaerts, P. E. M. Verheggen, J. Chromatogr.169 (1979), pp. 1-10 and R. L. Chien, D. S. Burgi, Anal. Chem. 64 (1992)pp. 489A-496A demonstrated that changes in electric field strengthbetween sample and background solution zones can focus (i.e., stack)charged species. In electrokinetic chromatography, J. P. Quirino, S.Terabe, Science, 282 (1998) pp. 465-68 and J. P. Quirino, S. Terabe,Anal. Chem. 71(8) (1999) pp. 1638-44 have shown that micelles can act toconcentration (i.e., sweep) neutral and charged species.

[0005] In CEC using particle (e.g., octadecyl silica) packed columns,focusing effects similar to that in gradient high performance liquidchromatography have been reported. These focusing effects were achievedusing (1) step-gradient elution, (2) preparation of the sample in anoneluting solvent, or (3) injection of a water plug after sampleinjection. M. R. Taylor, P. Teale, D. Westwood, D. Perrett, Anal. Chem.69 (1997) pp. 2554-58 were the first to report the use of astep-gradient for the preconcentration of steroidal samples in 1997. D.A. Stead, R. G. Reid, R. B. Taylor, J. Chromatogr. A 798 (1998) pp.259-67 achieved a 17-fold increase in the detection sensitivity of amixture of steroids by preconcentration using a noneluting samplematrix. Y. Zhang, J. Zhu, L. Zhang, W. Zhang, Anal. Chem. 72 (2000) pp.5744-47 also used a noneluting solvent for the preconcentration ofbenzoin and mephenytoin by a factor of 134 and 219, respectively. C. M.Yang, Z. El Rassi, Electrophoresis 20 (1999) pp. 2337-42 reported on thepreconcentration of a dilute sample of pesticides using a short plug ofwater injected after a long plug of sample. M. J. Hilhorst, G. W.Somsen, G. J. de Jong, Chromatographia 53 (2001) pp. 190-96 demonstratedpreconcentration of structurally related steroids using a nonelutingmatrix and step-gradient elution. A gain in sensitivity of 7 to 9 timeswas reported. Similarly, T. Tegeler, Z. El Rassi, Anal. Chem. 73(14)(2001) pp. 3365-72 just recently reported preconcentration of analytesin a mixture of carbamate insecticides using a combination of anoneluting matrix and step-gradient elution. The maximum allowablesample plug length was ˜20 cm and a 500-fold sensitivity increase isachieved for carbofuran. A further increase in detection sensitivity wasachieved by Zhang co-workers, who combined field-enhanced sampleinjection with solvent gradient elution. They demonstrated a 17,000-foldincrease in peak height for a positively charged analyte, propatenene.

[0006] It is desirable to provide a separation column with improvedcharacteristics and that is easy to make.

SUMMARY OF THE INVENTION

[0007] A separation column includes a separation channel and a fritlessseparation medium in the channel. The medium includes a porous matrix,and the porous matrix includes a metal organic polymer. The polymer maybe a photopolymer. In the preferred embodiment, the porous matrixcontains no chromatographic particles. The porous matrix may be used toboth preconcentrate and separate analytes without chromatographicparticles. The separation column allows for the concentration andseparation of larger volumes of analytes than a separation column withchromatographic particles.

[0008] A method of preparing a monolith is provided. A separation columnis provided. A mixture is introduced into the separation column. Themixture includes a metal organic compound. The mixture is irradiated toform a solid, porous matrix via photoinitiated polymerization. Thus, afritless separation medium is formed in the separation column. In thepreferred embodiment, the porous matrix contains no chromatographicparticles. Preparation of a fritless separation medium withoutchromatographic particles is simpler than preparing a separation mediumwith a frit or chromatographic particles. The porous matrix may be usedboth to preconcentrate and separate analytes.

[0009] The photochemical route to the preparation of the porous matrixhas many advantages: (1) short preparation time, (2) control of the poresize, (3) control over the placement and length of the porous matrix,(4) high mechanical strength, and (5) avoidance of high temperaturesthat lead to cracking.

[0010] A method of separating a sample of analytes is provided. Aseparation column including a separation channel and a fritlessseparation medium in the channel is provided. The medium includes aporous matrix, and the porous matrix includes a metal organic polymer,such as a photopolymer. In the preferred embodiment, the porous matrixcontains no chromatographic particles. A sample of analytes carried in asolution is introduced through the column. The medium concentrates theanalytes on the column. A solution is caused to flow through the column,thereby separating and eluting the analytes. The medium bothpreconcentrates and separates the analytes. In addition to the effectexerted by the medium, preconcentration can be further enhanced by asolvent gradient or sample stacking.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The above and other features and aspects of the present inventionwill become more apparent upon reading the following detaileddescription in conjunction with the accompanying drawings, in which:

[0012]FIG. 1 is a schematic longitudinal view of a separation column,according to an embodiment of the present invention;

[0013]FIGS. 2A and 2B are two representative electrochromatogramsshowing a plot of absorbance versus retention time for (a) one columnand (b) another column, using an embodiment of the present invention;

[0014]FIG. 3 is a representative electrochromatogram showing a plot ofabsorbance versus retention time using an embodiment of the presentinvention;

[0015]FIGS. 4A and 4B are SEM micrographs of embodiments of the presentinvention;

[0016]FIGS. 5A and 5B are representative electrochromatograms showingplots of absorbance versus retention time for different analytes usingembodiments of the present invention;

[0017]FIG. 6 is a representative electrochromatogram showing a plot ofabsorbance versus retention time using an embodiment of the presentinvention;

[0018]FIG. 7 (panels a and b) are representative electrochromatogramsshowing plots of absorbance versus retention time using an embodiment ofthe present invention;

[0019]FIG. 8 (panels a, b, and c) are representativeelectrochromatograms showing plots of absorbance versus retention timeusing an embodiment of the present invention;

[0020]FIG. 9 (panels a, b, c, d, and e) are representativeelectrochromatograms showing plots of absorbance versus retention timeusing an embodiment of the present invention;

[0021]FIG. 10 is a graphical representation showing a plot of thelogarithm of peak height ratio versus the percentage of water in asample using an embodiment of the present invention;

[0022]FIG. 11 (panels a, b, and c) are representativeelectrochromatograms showing plots of absorbance versus retention timeusing an embodiment of the present invention;

[0023]FIGS. 12A, 12B, and 12C are representative electrochromatogramsshowing plots of absorbance versus retention time using an embodiment ofthe present invention;

[0024]FIG. 13 (panels a and b) are representative electrochromatogramsshowing plots of absorbance versus retention time using an embodiment ofthe present invention;

[0025]FIG. 14 (panels a, b, c, d, and e) are representativeelectrochromatograms showing plots of absorbance versus retention timeusing an embodiment of the present invention;

[0026]FIG. 15 (panels a, b, c, d, e, and f) are representativeelectrochromatograms showing plots of absorbance versus retention timeusing an embodiment of the present invention;

[0027]FIG. 16 (panels a and b) are representative electrochromatogramsshowing plots of absorbance versus retention time using an embodiment ofthe present invention; and

[0028]FIG. 17 (panels a, b, c, d, and e) are representativeelectrochromatograms showing plots of absorbance versus retention timeusing an embodiment of the present invention.

[0029] For simplicity of description, like reference symbols are usedfor like or similar parts in this application.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030]FIG. 1 is a longitudinal cross-sectional view of a separationcolumn, according to an embodiment of the present invention. Theseparation column 11 includes a separation channel 13 in tube 12 and aseparation medium 15 in the channel 13. The separation column 11 may bea capillary or a planar chip, and the separation channel 13 may includea detection window. The separation medium 15 fills at least a section ofthe channel 13. The medium 15 is homogeneous and preferably does nothave frits or chromatographic particles. The medium 15 may be attachedto a channel wall 17 of the channel 13. Preferably, the medium 15 may becovalently bound to the channel wall 17.

[0031] Tube 12 can have many different cross-sections, such as acircular cross-section. Alternatively, tube 12 can have an elongatedcross-section. These and other cross-sections are possible for tube 12and are within the scope of the invention. Tube 12 may be a roundcapillary typically made of fused silica. The inside diameter (i.d.) ofthe capillary may be from about 10 μm to about 1000 μm, preferably fromabout 75 μm to about 500 μm. Tube 12 may be a planar chip or a confinedspace, such as a column confined by two sheets.

[0032] The medium 15 includes porous matrix, and the porous matrixincludes a metal organic polymer, such as a photopolymer. A precursor ofthe polymer may be a metal alkoxide, such as a silane. The metal may bealuminum, barium, antimony, calcium, chromium, copper, erbium,germanium, iron, lead, lithium, phosphorus, potassium, silicon,tantalum, tin, titanium, vanadium, zinc, or zirconium. The precursor mayinclude a photoactive group, such as methacrylate. In one embodiment,the precursor may be trimethoxysilypropyl methacrylate, also known asmethacryloxypropyltrimethoxy silane.

[0033] Different functionalized or derivatized monomers can be used toprepare a porous matrix 15 with different physical properties, such aspore size, polymer charge density, and hydrophobicity. Control of thepore shapes and sizes through the use of different porogens can resultin a porous matrix with a wide distribution of pore sizes (i.e.,pore-size gradient). A porous matrix with a pore-size gradient canfunction as “molecule sorter” in capillary electrophoresis and capillaryelectrochromatography. The porous matrix 15 can separate a mixture oflarge molecules whose size structures or chemistries (e.g., DNAfragments) may differ. In addition, separation columns 11 can bedesigned for reversed-phase, size-exclusion, affinity, ion-exchangechromatographies, etc.

[0034] A porous matrix 15 may be a mixed phase porous matrix preparedfrom a mixture of monomers. For example, the monomers may includemethacryloxypropyltrimethoxy silane, bis(triethoxysilyl)ethane, andbis(triethoxysilyl)octane. The mixed phase porous matrix may havedifferent properties, such as hydrophobicity.

[0035] The porous matrix has an affinity for analytes and can be used toboth preconcentrate and separate a sample of analytes. The affinity foran analyte may be described by the retention factor, k, of the analyte.The retention factor, k, is equal to $\begin{matrix}{k = \frac{{amount}\quad {of}\quad {component}\quad {in}\quad {stationary}\quad {phase}}{{amount}\quad {of}\quad {component}\quad {in}\quad {mobile}\quad {phase}}} & (1)\end{matrix}$

[0036] The retention factor, k, may also be expressed as $\begin{matrix}{k = \frac{t_{R} - t_{O}}{t_{O}}} & (2)\end{matrix}$

[0037] where t_(R) is the migration time of the analyte, and to is themigration time of an “unretained” analyte. The retention factor isaffected by the nature of the solvent, the nature of the analyte, thelength of the column, the permeability of the porous matrix, thehydrophobicity of the porous matrix, and the detailed morphology of theporous matrix.

[0038] The separation column 11 may be used for analytic orsemipreparative work. Separation of analytes in the submilligram tomilligram quantities may become possible with preconcentration onseparation columns 11. For example, more than about 100-nL of samplesolution at analyte concentrations in the mM levels can be injected intothe column without significant evidence of overloading.

[0039] A method of preparing a monolith in the separation column 11 isprovided. A separation column is provided. The separation column may bea round capillary typically made of fused-silica. The inside diameter(i.d.) of the capillary may be from about 10 μm to about 1000 μm,preferably from about 75 μm to about 500 μm.

[0040] A mixture that includes a metal organic compound is introducedinto the separation column. The mixture forms a solid, porous matrix viaphotoinitiated polymerization. The mixture may comprise a metal organicmonomer, a porogen, and a photoinitiator. The metal organic monomer maybe a metal alkoxide, such as a silane, or a mixture of metal alkoxides.The metal may be aluminum, barium, antimony, calcium, chromium, copper,erbium, germanium, iron, lead, lithium, phosphorus, potassium, silicon,tantalum, tin, titanium, vanadium, zinc, or zirconium. The metalalkoxide may include a photoactive group, such as methacrylate. In oneembodiment, the precursor may be trimethoxysilypropyl methacrylate, alsoknown as methacryloxypropyltrimethoxy silane. In another embodiment, theprecursor may be a mixture of methacryloxypropyltrimethoxy silane andanother precursor, such as bis(triethoxysilyl)ethane orbis(triethoxysilyl)octane.

[0041] The metal organic monomer may be added to an acid or basecatalyst for the hydrolysis of the precursor. The catalyst converts thealkoxy groups to hydroxyl groups. For example, a silane may undergo thefollowing hydrolysis reaction to form a fully hydrolyzed silane:

Si(OR)₄+4H₂O→Si(OH)₄+4ROH  (3)

[0042] The hydrolysis reaction may stop at a partially hydrolyzedsilane, Si(OR)_(4−n)(OH)_(n). The metal organic monomer and the catalystmay be stirred for about zero minutes to about twenty-four hours.

[0043] A porogen or a mixture of porogens may be mixed with the metalorganic monomer and the catalyst, and the mixture may be stirred forabout zero minutes to about twenty-four hours. During this time, themetal organic monomer may undergo a condensation reaction to formdimers, trimers, and other oligomers. For example, a partiallyhydrolyzed silane may undergo the following condensation reaction:

2(RO)₃SiOH→(RO)₃Si—O—Si(OR)₃+H₂O  (4)

[0044] Larger oligomers may be formed by increasing the temperature ofthe reaction.

[0045] The porogen provides a molecular template to form pores withinthe matrix. For example, the porogen may be a solvent, such as tolueneor a 1:1 mixture of hexane and toluene, a polymer, or an inorganic salt,such as sodium chloride powder or sodium sulfate. The polymeric porogenmay be poly(methyl methacrylate) or polystyrene, as reported by D.Horak, J. Labsky, J. Pilar, M. Bleha, Z. Pelzbauer, F. Svec, Polymer34(16) (1993) pp. 3481-89. The porogen may be selected controllably toform pores in the matrix 15. The porosity of the matrix may becontrolled by the type of chemical (i.e., porogen) used and its volumeor concentration in the reaction solution. For example, a molar orvolume ratio of monomer to porogen may be selected to form pores in themixture. By adjusting the molar ratio of the monomer and porogen, thephysical properties (e.g., pore sizes) of the matrix may be controlled.

[0046] The photoinitator or a photoactive group, such as methacrylate,on the monomer absorbs light to catalyze the polymerization of the metalorganic compound. In one embodiment, the photoinitiator may be Irgacure1800, available from Ciba Geigy, Tarrytown, N.Y.

[0047] If the separation column has an outer coating that is nottransparent to the light source, the coating is first removed to make anirradiation window. The length of the coating will determine the lengthof the porous matrix formed within the separation column.

[0048] The mixture is introduced into the separation column. Forexample, the mixture may be flowed through the separation column using asyringe. The ends of the separation column may be sealed.

[0049] The mixture is irradiated, thereby forming a fritless,homogeneous separation medium in the separation column. For example, theseparation column may be exposed to radiation for a short period oftime, such as about five minutes. The wavelength of the radiation isdependent on the type of photoinitiator or photoactive group used in thereaction. The radiation may include visible or ultraviolet light. Forexample, radiation of 365 μm may be used for the photoinitiator Irgacure1800. The photoactive group methacrylate may be photopolymerized at awavelength of 300 nm or 365 nm, as reported in C. Yu, F. Svec, J. M. J.Frechet, Electrophoresis 21(1) (2000) pp. 120-27 and H.-G. Woo, L.-Y.Hong, S.-Y. Kim, S.-H. Park, S.-J. Song, H.-S. Ham, Bull. Korean Chem.Soc. 16 (1995) pp. 1056-59, respectively.

[0050] A photochemical reaction occurs when the mixture is exposed toradiation. The photoinitator or photoactive group on the monomer absorbsthe light to catalyze the polymerization of the metal organic compound.

[0051] The photochemical route to the preparation of a porous matrix hasmany advantages: (1) short preparation time, (2) control of the poresize, (3) control over the placement and length of the porous matrix,(4) high mechanical strength, and (5) avoidance of high temperaturesthat lead to cracking.

[0052] The separation medium preferably does not have frits orchromatographic particles, so the preparation of the separation mediumis easier than preparation of a conventional separation medium withfrits or chromatographic particles is.

[0053] Optionally, an organic solvent may be introduced into theseparation column. For example, the organic solvent may be ethanol. Theporous matrix may be washed clean of any unreacted material, theporogen, and the photoinitiator using an organic solvent. The solventmay be flowed through the separation column using a syringe or othermeans.

[0054] The separation column may be conditioned with a separationsolution before using the column for separation. A separation solutionmay comprise a buffer, such as aqueous ammonium acetate, and an elutingsolvent, such as acetonitrile.

[0055] A method to separate a sample of analytes is provided. A sampleof analytes is introduced in a sample solution through a separationcolumn 11. Analytes includes neutral species, such as polycyclicaromatic hydrocarbons, alkyl benzenes, alkyl phenyl ketones, andsteroids, and charged species, such as peptides. The sample solution maycomprise a buffer, such as aqueous ammonium acetate, and an elutingsolvent, such as acetonitrile.

[0056] The separation column 11 includes a separation channel 13 and afritless, homogeneous separation medium 15 in the channel 13. Theseparation column 11 may be a capillary or planar structure. Theseparation medium 15 preferably does not have frits or chromatographicparticles. The use of a homogeneous separation medium is advantageousbecause, in some applications, the use of chromatographic particles(i.e., inhomogeneous separation phase) introduces unwanted broadening(i.e., lack of resolution). The separation medium 15 may possiblyinclude two sections containing substantially the same poroushomogeneous stationary phase, and the two sections may be separated byanother section, such as a monolith with a different pore size orsurface charge. Preferably, the separation medium 15 is continuous.

[0057] The separation medium 15 includes a metal organic photopolymerand concentrates the analytes on the column. The sample may beintroduced by applying a pressure or a voltage to the column 11. Forexample, the pressure may be 0.5 psi or 20 psi for a period of time,such as two to 1920 seconds, or a field strength of about 40 V/cm may beapplied for a period of time. For example, an injection plug length ofgreater than about two centimeters may be injected into the column 11.

[0058] The analyte is concentrated by the column 11. The extent ofpreconcentration is purely dependent on the retention factor, the kvalue. The retention factor is affected by the nature of the solvent,the nature of the analyte, and the detailed morphology of the separationmedium. Moreover, the flow rate hardly influenced the extent ofpreconcentration.

[0059] The highly porous nature of the porous matrix results in a highmass transfer rate for the analyte, which facilitates thepreconcentration effect. The high mass transfer rates arise from theenhanced accessibility of the analytes to the binding sites of theporous structure. Because of the high mass transfer rates, the kineticsof analyte-porous matrix interaction (i.e., the partitioning of theanalyte between the mobile and stationary phases) is not therate-limiting step in the separation. The high mass transfer ratedistinguishes this separation method from previous forms ofchromatographic separations. With this separation method, because of thehigh mass transfer rate, it is possible to inject and concentrate largervolumes of sample solution than in columns containing normalchromatographic materials.

[0060] The total preconcentration effect is directly proportional to theretention factor, with longer injection plug lengths (e.g., greater thanabout 25 mm) leading to severe peak broadening of analytes having low-kvalues. This behavior implies a maximum length of sample plug for eachanalyte before peak shape becomes compromised.

[0061] A major advantage of on-line preconcentration is that it lowersthe detection limit for a given analyte. Another advantage is thatpreconcentration may be used to clean up the analytes from possibleinterfering species found in the sample matrix.

[0062] A separation solution is caused to flow through the column 11,thereby separating and eluting the analytes. The separation solution maybe caused to flow by applying a voltage or a pressure to the column 11.For example, the pressure may be 0.5 psi or 20 psi for a period of time,such as two to 1920 seconds, or a field strength of 300 V/cm may beapplied for a period of time. The separation solution may comprise abuffer, such as aqueous ammonium acetate, and an eluting solvent, suchas acetonitrile. In one embodiment, the separation solution is the sameas the sample solution.

[0063] The porous matrix acts to extract the analytes from solution aswell as provides the stationary phase for chromatographic separation ofthe analytes. It is this extractor-separator combination that gives thismethod such power. For example, more than about two-centimeter plugs ofsample solution can be loaded into the capillary and concentrated usinga separation solution that is the same as the sample solution.

[0064] In one embodiment, in addition to the effect exerted by theporous matrix, a solvent gradient may be used to further enhancepreconcentration of the analytes. In this embodiment, the sample may bedissolved in a solution with a higher concentration of a buffer (e.g.,water) than in the separation solution. The higher concentration of thebuffer in the sample solution increases the affinity of the sample tothe stationary phase. When a solvent gradient is used, the plug lengthcan be longer than the length of the column. For example, injection of a91.2-cm plug, which was more than three times the total length of thecapillary, was possible with only a minor loss in resolution.Improvements in peak heights obtained under gradient conditions can bemore than a thousand-fold.

[0065] For neutral analytes, two approaches exist for using gradients onthe porous matrix. The first approach is to increase the organic solventratio between the separation solution and the sample solution. Thesecond approach is to increase the retention factor k in the separationby increasing the percentage of water in the separation solution whilemaintaining a reasonable percentage of organic solvent between theseparation solution and the sample solution. Analysis is faster with thefirst approach, whereas the resolution is better with the second one.

[0066] In another embodiment of the present invention, in addition tothe effect exerted by the porous matrix, sample stacking may be used tofurther enhance the preconcentration of analytes. Sample stacking is thefocusing of charged analytes when analytes pass the concentrationboundary that separates regions of high and low electric fieldstrengths. The high electric field zone is a lower conductivity samplesolution containing more of the eluting solvent, whereas the lowelectric field region is a higher conductivity separation solution. Theeluting solvent, such as acetonitrile, has a lower conductivity than thebuffer, such as aqueous ammonium acetate. Thus, a higher concentrationof the eluting solvent results in lowering the sample matrixconductivity.

[0067] The separation column is prepared with the separation solution.When analytes are introduced into the separation column and a voltage isapplied, the analytes in the sample solution at the inlet of the columnrapidly accelerate toward the separation solution (lower electric fieldstrength) already in the column, where on crossing the boundary betweenthe sample solution and the separation solution, they slow down andstack into narrow zones at the interface.

[0068] Sample stacking is basically caused by the change inelectrophoretic velocity at the concentration boundary. Electrophoreticvelocity is the product of electrophoretic mobility and electric fieldstrength. Focusing occurs (sample stacking) when the electrophoreticvelocity decreases at the concentration boundary. Sample stacking isalso explained using the fundamentals of isotachophoresis and Kohlrauschrules.

[0069] There are two approaches to perform sample stacking on a porousmatrix. The first approach is to increase the percentage of organicsolvent, such as acetonitrile. The second is to decrease theconcentration of the buffer component in the sample solution. Increasingthe percentage of acetonitrile or other suitable organic solvent isespecially useful for real samples containing high concentration ofsalts. Desalting, for example by dialysis, is therefore not necessary tomake a lower conductivity solution for injection. Use of organicsolvents is also useful for biological samples when deproteination ispart of the sample preparation.

[0070] The invention is described in more detail by the way of thefollowing examples. The following examples are presented solely for thepurpose of further illustrating and disclosing the present invention,and are not to be construed as limiting the invention.

EXAMPLE 1

[0071] Materials and Chemicals. Fused-silica capillaries (75-μmi.d.×365-μm o.d.) were purchased from Polymicro Technologies, Phoenix,Ariz. Methacryloxypropyltrimetoxysilane (MPTMS) was purchased fromGelest, Tullytown, Pa. and Sigma-Aldrich, Milwaukee, Wis. and was usedwithout purification. HPLC-grade toluene, phenanthrene, pyrene, alkylbenzenes, alkyl phenyl ketones, and steroids were purchased fromSigma-Aldrich, Milwaukee, Wis. Irgacure 1800 was received from Ciba,Tarrytown, N.Y.

[0072] Instrumentation. A Beckman P/ACE 2000 capillary electrophoresisinstrument with a UV-absorbance detector was used to carry out all CECexperiments. An XL-1500 UV cross-linker, available from SpectronicsCorp., Westbury, N.Y., equipped with six 15 W blacklight tubes ofpredominantly 365-nm wavelength was used to irradiate the reactionsolutions. Scanning electron microscopy (SEM) analyses were performed ona Philips SEM 505 scanning electron microscope, available fromEindhoven, Netherlands.

[0073] Polymerization Procedure. The monomer stock solution was preparedjust prior to use by adding 375 μL of MPTMS to 100 μL of 0.12 N HCl.This solution was stirred at room temperature for approximately thirtyminutes to afford a clear, monophasic solution. An appropriate amount oftoluene (porogen) was added to the monomer stock solution, as shownbelow in Table 1. TABLE 1 Capillary Column % toluene (v/v) A 90 B 80 C75 D 73 E 65 F 50

[0074] The photoinitator, Irgacure 1800, was added first to the tolueneas 5% of the total weight of the toluene/monomer stock solution. Thisphotoinitator solution was then added to the corresponding amount ofmonomer stock solution, and stirred for thirty minutes at roomtemperature to afford a yellow, monophasic solution. To minimize theevaporation of toluene, the solution was prepared in a vial with apolysilicone cap through which the capillary was inserted during fillingwith the solution.

[0075] A 15-cm stripe of the polyimide coating on a 30-cm long capillarywas removed using a razor blade positioned at 45° to the capillarysurface. The mechanical stability of the capillary was remarkably gooddespite the removal of a stripe of polyimide coating. The irradiationlight entered the capillary only through this 15-cm stripe. No monolithwas formed in the capillary where the polyimide coating (“mask”)remained intact.

[0076] Using a 0.5-mL disposable syringe, approximately 0.2 mL of thereaction solution was flushed through the capillary to wet thoroughlythe wall surface before filling the capillary with the solution. Thisresulted in bonding of the monolith to the capillary wall. No specialpretreatment of the capillary wall was necessary to bond the monolith tothe wall. The filled capillaries were irradiated (900 mJ/cm²) in a UVcross-linker using 365-nm light for five minutes to form the porousmatrix.

[0077] After irradiation, the capillaries were washed with ethanol usinga hand-held syringe to removed unreacted reagents or porogens. Becausethe monoliths were highly permeable, high pressures were not required todrive liquid through the capillaries. Once the unreacted reagents wereremoved, the monolith became opaque and could be viewed clearly throughthe capillary without the aid of a microscope. The homogenity of theporous matrix was confirmed at 100× magnification. Burning off thepolyimide coating imediately after the monolith section with fumingsulfuric acid made a detection window.

[0078] Once fabricated, the capillary was successfully installed in thecartridge without any damage. The capillary was conditioned with theseparation buffer for approximately five minutes using a syringe and ahand-held vise. Once in the instrument, the capillary was furtherconditioned by pressure rinsing (20 psi) with the separation buffer orby electrokinetically conditioning at 5 kV or 10 kV for thirty minutes.

[0079] Characterization. SEM was used to study the morphology of theseparation column. A capillary was sectioned carefully to expose themonolith. The sectioned pieces of capillary were sputtered with goldprior to SEM analyses.

[0080] Analyte Separation. The analytes were prepared in the mobilephase to prevent gradient effects during the CEC experiments. The mobilephase was made up of various ratios (v/v) of 50 mM ammonium acetate,water, and acetonitrile. A new sample solution was used for everyinjection to maintain the same concentration of acetonitrile in thesample solution and the mobile phase.

[0081]FIG. 2A is a representative electrochromatogram showing a plot ofabsorbance versus retention time for column B. The separation wasperformed with a 50 mM ammonium acetate/water/acetronitrile (1/3/6)solution. The sample solution was injected at 0.5 psi pressure for threeseconds, and the separation was performed with an applied voltage of 1kV at a temperature of 20° C. and detected at 214 nm. The elution orderof the separation was (1) thiourea, (2) tetrahydrofuran, (3)naphthalene, (4) phenanthrene, (5) fluoranthene, (6) pyrene, (7)1,2-benzanthracene, and (8) 1,2,5,6-bienzanthracene.

[0082]FIG. 2B is a representative electrochromatogram showing a plot ofabsorbance versus retention time for column D. The separation wasperformed with a 50 mM ammonium acetate/water/acetronitrile (1/4/5)solution. The sample solution was injected at 0.5 psi pressure for threeseconds, and the separation was performed with an applied voltage of 15kV at a temperature of 20° C. and detected at 200 nm. The elution orderof the separation was (1) benzene, (2) toluene, (3) ethylene benzene,(4) propyl benzene, (5) butyl benzene, and (6) hexyl benzene.

[0083] The elution order of the column was similar to that ofreversed-phase chromatography with the larger molecular weight or morehydrophobic analytes eluting later than the smaller molecular weight ormore hydrophilic analytes. Elution of the analytes in both figuresoccurred in less than seven minutes. Bubble formation was not a problemduring the CEC experiments, for which the typical operating currentswere between 3 and 10 μA.

[0084] For a typical capillary column D, efficiencies of up to 100,000plates/m are achieved for thiourea, a less-retained compound. Smallvariations in the elution times were observed for thiourea (0.65% RSD),naphthalene (1.10% RSD), phenanthrene (1.14% RSD), and pyrene (1.14%RSD) over a period of three days (n=33).

[0085]FIG. 3 is a representative electrochromatogram showing a plot ofabsorbance versus retention time for column D. The separation wasperformed with a 50 mM ammonium acetate/water/acetronitrile (1/3/6)solution. The sample solution was injected at 0.5 psi pressure for threeseconds, and the separation was performed with an applied voltage of 1kV at a temperature of 20° C. and detected at 214 μm. A sample ofthiourea, napthalene, phenanthrene, and pyrene was separated within 110minutes at an applied pressure of only 20 psi (the maximum limit of theinstrument). Peak tailing was most severe for pyrene because of itsstrong interaction with the porous matrix, and tailing was not observedfor thiourea, which has low retention on the column.

[0086]FIG. 4A is a scanning electron micrograph of the cross-section ofa metal organic photpolymer formed with 80% (v/v) toluene (capillary B)in a 75-μm-i.d. capillary column. The micrograph showed aninterconnecting network of 1-μm spherical structures through whichmicrometer-sized macropores (as large as 5 μm) are interspersed.

[0087]FIG. 4B is a scanning electron micrograph of the cross-section ofa metal organic photpolymer formed with 50% (v/v) toluene in a75-μm-i.d. capillary column. In contrast to the porous matrix shown inFIG. 4A, the structure shown in FIG. 4B was a dense photopolymer withmacropores of 2 μm or less in diameter. Consequently, the matrix in FIG.4B was less permeable, and a significant back pressure occurs. No liquidcould be driven through the column at pressures near 200 psi.

[0088] The permeability of a porous matrix was determined by the linearvelocity of the porous matrix, which is proportional to permeability asdescribed in Darcy's law. The permeability of a porous matrix as afunction of the macropore size was highly dependent on the volume andtype of porogen used to prepare the photopolymer. For a column made with90% (v/v) toluene (column A), the linear velocity is 12.3 cm min, and an80% (v/v) column (column B) had a linear velocity of 3.3 cm/min, whereasa column made with 73% (v/v) toluene (column D) had a linear velocity of0.6 cm/min. These linear velocity data suggested that the macroporesdecrease with decreasing porogen concentration. This behavior wasconsistent with what has reported in the literature.

EXAMPLE 2

[0089] The separation column was prepared as described in Example 1. Amixture of 1:1 hexane/toluene was used for the porogen. The separationcolumn had a separation performance similar to that of a separationcolumn made with 80/20 toluene/reaction solution. A column efficiency of68,000 plates/m (RSD 7.0%, n=5) for thiourea and an electroosmotic flow(EOF) velocity of 3.7 cm/min was obtained.

EXAMPLE 3

[0090] A mixture of 375 μL of MPTMS and 100 μL of 0.12 M hydrochloricacid was stirred for thirty minutes at room temperature. 27 parts ofthis mixture was combined with 73 parts of toluene to give 200 μL of thefinal solution. 5% by weight of the final solution of the photoinitatorIrgacure 1800 was added, and the resulting sol-gel solution was stirredfor five minutes before use. A 75-μm i.d.×365-μm o.d. fused silicacapillary was filled with the sol-gel solution, and the separationcolumn was exposed to UV light in a Spectrolinker X1-1500 at 365 nm toaffect photopolymerization. The polymerization length of the porousmatrix was controlled by removing a 15-cm strip of the polyimide coatingof the capillary prior to irradiation for five minutes. Unreactedreagents were flushed with ethanol. The total length of the capillarywas 25.6 cm (18.8 cm from inlet to the detector window). The resultingcolumn was conditioned with the separation solution prior to use.

[0091] All electrophoresis experiments were performed with a BeckmanP/ACE 2000. The capillaries were thermostated at 20° C. Injections weredone using pressure (i.e., 0.5 psi and 20 psi) or voltage (1 kV to 10kV) and varied in duration from two seconds to 1920 seconds. Detectionwas done at 214 or 254 nm. Data analysis was performed with GRAMS/32version 4.02, available from Galactic Industries Corporation, Salem,N.H.

[0092]FIGS. 5A and 5B are representative electrochromatograms showingplots of absorbance versus retention time using an embodiment of thepresent invention. The figures illustrate the increase in detectionsensitivity with an increase in injected plug length in the CECseparation of a mixture containing the small molecule, thiourea, threepolycyclic aromatic hydrocarbons (PAHs), and eight alkyl phenyl ketones.To eliminate solvent gradient effects, the sample was prepared in theseparation solution. The sample and separation solutions were 50 mMammonium acetate/water/acetonitrile (1/4/5). In FIG. 5A, the pluglengths were 0.1 mm, 6.8 mm, 13.7 mm, 27.4 mm, and 34.2 mm. The 0.1 mmplug length was for an applied pressure of 0.5 psi, whereas all otherplug lengths were for an applied pressure of 20 psi. The applied voltagefor the separation was 20 kV, and the absorbance was measured at 214 nm.The elution order of the column was (1) 12.5 μM thiourea, (2) 51.0 μMnaphthalene, (3) 1.0 μM phenanthrene, and (4) 123 μM pyrene.

[0093] In FIG. 5B, the column was prepared in the same manner as thecolumn as described earlier, except that the column was post-modified bycontinuous flow of (3,3,3-trifluoropropyl)trichlorosilane for thirtyminutes at room temperature and followed by rinsing with toluene. Theplug lengths were 0.7 m, 7.2 mm, 10.7 mm, 17.9 mm, and 28.6 mm. Theapplied voltage was 15 kV, and the absorbance was measured at 254 nm.The elution order of the column was (1) 5 μM thiourea, (2) acetophenone,(3) propiophenone, (4) butyrophenone, (5) valerophenone, (6)hexanophenone, (7) heptanophenone, (8) octanophenone, and (9)decanophenone. The concentration of each of the alkyl phenyl ketones was0.1 μg/mL in the separation solution.

[0094] For the PAH mixture illustrated in FIG. 5A, the peaks were barelyvisible with the typical injection of a 0.1 mm plug length, but the peakheights increased when the plug length increased from 6.8 to 34.2 mm.Thus, the separation column, an embodiment of the present invention,allowed for the injection of a longer plug length than a typicalseparation column does. Similarly, for the alkyl phenyl ketone mixtureillustrated in FIG. 5B, the peak heights increased when the plug lengthwas increased from 0.7 mm to 57.3 mm. As the plug length was increased,all four peaks showed increased broadening, but the later eluting peaksare more symmetrical to a small extent than the earlier ones. Thisbehavior is backwards from what is observed in typical chromatographicseparations in which the later eluting peaks are less symmetrical thanthe earlier ones because of dispersion effects. These results suggestthat the analytes accumulate at the inlet of the poroux matrix duringthe injection, with the more retentive species being localized moreeffectively than the less retentive ones.

[0095] In FIG. 5A, the improvement in peak heights for a 27.4-mminjection compared to a typical injection of 0.1 mm is 50, 125, and 127times for naphthalene, phenanthrene, and pyrene, respectively. Thesample solution in the 27.4-mm injection is a 10-fold dilution of thesample in the typical 0.1-mm injection.

EXAMPLE 4

[0096] The separation column was prepared as described in Example 3. Thesample and separation solution was 50 mM ammoniumacetate/water/acetonitrile (1/3/6). The samples were injected at 1 kV.The applied voltage was 15 kV, and the absorbance was detected at 214nm. FIG. 6 is a representative electrochromatogram of a plot showingabsorbance versus retention time using an embodiment of the presentinvention. 39.0 mM of naphthalene in the separation solution wasinjected for five seconds, as represented by signal a, and a 3.9 mM ofnaphthalene in the separation solution was injected for eighty-fiveseconds, as represented by signal b. The corrected peak areas (peakarea/migration time) for both electrochromatograms were made close toeach other by controlling the injection time of the ten-fold dilution ofsample. The corrected peak areas of the electropherogram in lines a andb are 0.0023 (% RSD=0.02%, n=3) and 0.0025 (% RSD=0.00, n=3) arbitraryunits/min, respectively. This comparison was done such that the amountof naphthalene molecules injected for each run is the same.

[0097] Preconcentration was evidenced by the slightly higher peak heightfor the longer injection of diluted sample and almost the same correctedpeak widths (peak width/migration time) for both experiments, despitethe different sample concentration. The peak heights of theelectrochromatograms in signals a and b were 0.0869 (% RSD=0.36%, n=3)and 0.0937 (% RSD=0.06%, n=3) arbitrary units, respectively. The peakwidths of the electrochromatograms in signals a and b were 0.0253 (%RSD=0.07%, n=3) and 0.0249 (% RSD=0.01%, n=3) arbitrary units/min,respectively. The shift in migration time on line b was caused by thelonger injection time, which made the center of the sample plug closerto the detector window.

EXAMPLE 5

[0098] A mixture of 575 μL of MPTMS and 100 μL of 0.12 M hydrochloricacid was stirred for thirty minutes at room temperature. 20 parts ofthis mixture was combined with 80 parts of toluene to give 200 μL of thefinal solution. The photoinitiator was added as 10% of the total volumeof the final solution, and the resulting sol-gel solution was stirredfor five minutes before use. The separation column was prepared asdescribed above in Example 3. Unreacted reagents were flushed withtoluene. The surface of the porous matrix was modified by continuousflow of pentafluorophenyltrichlorosilane through the capillary forforty-five minutes at room temperature and followed by rinsing withtoluene.

[0099]FIG. 7 (panels a and b) are representative electrochromatogramsshowing plots of absorbance versus retention time using an embodiment ofthe present invention. The separation solution was 50 mM phosphoricacid/water/acetonitrile (1/5/4). The applied voltage was 15 kV, and theabsorbance was detected at 214 nm. FIG. 7 (panel a) shows a 0.5 psiinjection at 0.1 mm plug length of test peptides, and FIG. 7 (panel b)shows a 0.5 psi injection at 12 mm plug length of test peptides. Thetest peptides, which were charged analytes, were (1) bradykinin, (2)angiotensin II, (3) tripeptide I, (4) tripeptide II, and (5) methionineenkephalin. The concentration of the peptides were 16.7 μg/ml. Thecathode directed velocities of the peptides were dictated by bothelectrophoretic and electroosmotic flow effects. The peptides had a netpositive charge at the pH of the separation solution (pH=2). Theimprovement in peak heights for the longer injection compared to atypical injection of 0.1 mm plug length is 21, 19, 16, 18, and 22 timesfor bradykinin, angiotensis II, tripeptide I, tripeptide II, andmethionine enkephalin, respectively. This result demonstrates theusefulness of this method for charged analytes.

EXAMPLE 6

[0100] The separation column was prepared as in Example 3. FIG. 8(panels a, b, and c) are representative electrochromatograms showingplots of absorbance versus retention time using an embodiment of thepresent invention. FIG. 8 shows an analysis of a urine sample, spikedwith 0.1 mM hydrocortisone (peak 1), 0.3 mM progesterone (peak 2) and0.2 mM cortisone (peak 3). Four parts of spike or unspiked urine wasmixed with six parts of acetonitrile and centrifuged to remove proteins.One part of each supernatant was mixed with one part of 50 mM ammoniumacetate/water/acetonitrile (1/7/2) before injection. FIG. 8 (panel a)shows an injection plug length of 0.1 mm, and FIG. 8 (panel b) shows aninjection plug length of 21.4 mm. FIG. 8 (panel c) represents a 21.4-mminjection plug length of urine blank. The separation solution consistedof 50 mM ammonium acetate/water/acetonitrile (1/5/4). The appliedvoltage for the separation was 17 kV, and the absorbance was measured at254 mn. After protein precipitation with acetonitrile, these steroidswere detected and quantified with a 21.4-mm injection of the samplesolution, but are weakly detected with a typical 0.1-mm injection. Acomparison between the blank run and the spiked run showed that thesample matrix, which still contained other biomolecules, did notsignificantly interfere with steroid analysis on the separation column.This result demonstrates the usefulness of the technique for biofluidanalysis.

EXAMPLE 7

[0101] The separation column was prepared as described in Example 3.FIG. 9 (panels a, b, c, d, and e) are representativeelectrochromatograms showing plots of absorbance versus retention timeusing an embodiment of the present invention. Sample plug lengths of 1.1cm were injected. The separation solution was 5 mM ammonium acetate in60% acetonitrile, and the sample solutions were 5 mM ammonium acetate in60% acetonitrile (panel a), 50% acetonitrile (panel b), 40% acetonitrile(panel c), 30% acetonitrile (panel d), and 20% acetonitrile (panel e).The applied voltage for the separation was 15 kV, and the absorbance wasdetected at 254 nm. The elution order was (1) thiourea, (2)acetophenone, (3) propiophenone, (4) butyrophenone, (5) valerophenone,(6) hexanophenone, (7) heptanophenone, (8) octanophenone, and (9)decanophenone. The concentration of each analyte was 2 nl/ml.

[0102] The retention factors, k, obtained for acetophenone (peak 2),propiophenone (peak 3), butyrophenone (peak 4), valerophenone (peak 5),hexanophenone (peak 6), heptanophenone (peak 7), octanophenone (peak 8),and decanophenone (peak 9) were 0.18, 0.25, 0.32, 0.41, 0.53, 0.67,0.85, and 1.33, respectively. In this study, thiourea (peak 1) was usedas the essentially unretained neutral solute for the determination of k.The value of k and migration time follows the increase in alkyl chainlength. In general, the peak shapes and resolution improved when thewater concentration was increased from 40% to 50%, 60%, 70%, and 80%, asevidenced by panels a, b, c, d, and e, respectively.

EXAMPLE 8

[0103] The experimentation conditions were the same as in Example 7.FIG. 10 is a graphical representation of a plot showing the logarithm ofpeak height ratio (peak height obtained from a higher concentration ofwater in the sample matrix divided by peak height obtained from a samplematrix similar to that of the separation solution) versus the percentageof water in the sample matrix. The data indicated that a limit exists towhich the peak heights can be improved by increasing the concentrationof the buffer in the sample matrix. Preconcentration was improved owingto the increased attraction of the analytes to the porous matrix. Whenthe value for the logarithm of the peak height ratio was less than 1,about 1, or greater than 1 there was a decrease, no change, or increase,respectively, in peak height compared to a similar injection using theseparation solution as the sample matrix. For all test APKs, peakheights improved when the water concentration was increased from 40% to50% and from 50% to 60%. Peak heights did not improve when thepercentage of water was increased from 60% to 70% or more, except forthe two lowest k analytes (acetophenone and propiophenone) when thepercentage of water was increased from 60% to 70%. Peak heights worsenedfor the higher k analytes (heptanophenone, octanophenone, anddecanophenone) in the 80% water matrix. The reason for the decrease inpeak heights is the decrease in the solubility of the high k analytes inthe highly aqueous sample matrix. The corrected peak areas, which is ameasure of the amount of sample loaded for octanophenone anddecanophenone is 10% to 60% lower in the 80% water matrix compared tothe other sample matrices used. To avoid solubility problems, the testAPKs in succeeding experiments were prepared in matrices having at least30% acetonitrile.

EXAMPLE 9

[0104] The separation column was prepared as described above in Example3. FIG. 11 (panels a, b, and c) are representative electrochromatogramsshowing plots of absorbance versus retention time using an embodiment ofthe present invention. The separation solution is 5 mM ammonium acetatein 60% acetonitrile. The plug lengths were 0.2 cm for a sample solutionof 5 mM ammonium acetate in 60% acetonitrile (panel a); 2.74 cm for thesame sample solution was used in panel a (panel b), and 2.74 cm for asample solution of 5 mM ammonium acetate in 30% acetonitrile (panel c).Other conditions and identification of peaks are the same as in Example7.

[0105] The gradient condition, as shown in FIG. 11 (panel c), showedimproved resolution and peak shapes. Improvements in peak heights underthe gradient condition illustrated in FIG. 11 (panel c) were 36, 35, 38,41, 42, 38, 32, and 24 times for acetophenone, propiophenone,butyrophenone, valerophenone, hexanophenone, heptanophenone,octanophenone, and decanophenone, respectively. The % RSDs (n=5) ofmeasured peak heights ranged from 0.9% to 2.5%. % RSDs (n=5) ofmigration time ranged from 0.3% to 0.5%. The procedure is thereforereproducible in a single column.

EXAMPLE 10

[0106]FIGS. 12A, 12B, and 12C are representative electrochromatogramsshowing plots of absorbance versus retention time using an embodiment ofthe present invention. The plug lengths were 2.74 cm for sample solutionof 5 mM ammonium acetate in 40% acetonitrile (FIG. 12A), 2.74 cm forsample solution of 5 mM ammonium acetate in 30% acetonitrile (FIG. 12B),and 5.48 cm for a sample solution the same as in FIG. 12B (FIG. 12C).The separation solutions were 5 mM ammonium acetate in 60% acetonitrile(FIG. 12A) and 5 mM ammonium acetate in 50% acetonitrile (FIGS. 12B and12C). Other conditions and identification of peaks are the same as inExample 7.

[0107] The k values were higher in FIG. 12B than in FIG. 12A because ofthe high percentage of water in the separation solution. The analytemolecules were more attracted to the PSG phase at high percentages ofwater. The distribution constant K (number of moles of solute in the PSGphase divided by the number of moles of solute in the separationsolution), which is directly proportional to k increases with increasingconcentration of water in the separation solution. In FIG. 12B, the kvalues for acetophenone, propiophenone, butyrophenone, valerophenone,hexanophenone, heptanophenone, octanophenone, and decanophenone were0.29, 0.47, 0.65, 0.92, 1.28, 1.76, 2.37, and 4.25, respectively. Tomaintain the gradient effect constant, the percentage of organic solventratio between the separation solution and sample matrix was kept at thesame value for FIGS. 12A and 12B. For reasons still unknown, the resultin FIG. 12B shows that for analytes with lower k values (acetophenoneand propiophenone) there were slight increases in peak heights comparedto FIG. 12A. For the other test solutes, there are some decreases inpeak heights.

[0108]FIG. 12C illustrates what happens for a longer injection plug of5.48 cm and a higher percentage of water in the separation solution.Improvements in peak heights were 31, 33, 55, 44, 44, 37, 29, and 19times for acetophenone, propiophenone, butyrophenone, valerophenone,hexanophenone, heptanophenone, octanophenone, and decanophenone,respectively. As in FIG. 11 (panel c), the improvements in peak heightsdo not follow k, unlike in nongradient conditions. The improvements inpeak heights were comparable to those obtained with a higher percentageof organic solvent between the separation solution and the sample matrix(FIG. 11, panel c). Note that the injection plug is two times shorter inFIG. 11 (panel c) than in FIG. 12C.

EXAMPLE 11

[0109]FIG. 13 (panels a and b) are representative electrochromatogramsshowing plots of absorbance versus retention time using an embodiment ofthe present invention. The plug lengths were 0.22 mm (panel a) and 19.5cm (panel b). The separation solution was 5 mM ammonium acetate in 60%acetonitrile. The sample solutions were: 5 mM ammonium acetate in 60%acetonitrile (panel a) and 36% acetonitrile (panel b). The sampleconcentrations were 11 to 53 μg/ml (panel a) and 1.1 to 5.3 μg/ml (panelb). The applied voltage was 30 kV, and the absorbance was detected at214 nm. The elution order was thiourea (peak 1), naphthalene (peak 2),phenanthrene (peak 3), pyrene (peak 4), and benz(e)acephenanthylene(peak 5).

[0110] A solvent gradient improved detection of four PAHs, as shown inFIG. 13 (panel b). A high percentage of acetonitrile (60%) in theseparation solution, a shorter PSG length (10 cm), and a highelectric-field strength (781.3 V/cm) were used for faster analysistimes. FIG. 13 (panel a) is a 0.22-mm typical injection of sampleprepared in the separation solution. FIG. 13 (panel b) is a 19.5-cminjection using a gradient where the sample is in a 36% acetonitrilematrix, which provides a high percentage of organic solvent between thesample solution and the separation solution. Longer than 19.5-cm plugcause broadening of the naphthalene peak. It is interesting to note thatthe injection length is longer than the length from the inlet to thedetector window (18.8-cm). The faster eluting thiourea zone is actuallyobserved during the sample injection. The thiourea zone is therefore atthe detection window at the start of the separation voltage.

[0111] Improvements in peak heights for naphthalene (peak 2),phenanthrene (peak 3), pyrene (peak 4), and benz(e)acephenanthylene(peak 5) are 346, 437, 409, and 315 times, respectively. The sampleconcentrations in FIG. 13 (panel b) were 10-fold lower than in FIG. 13(panel a). For naphthalene, phenanthrene, and pyrene, the values statedabove are 6.9, 3.5, and 3.2 times better than that previously reportedunder nongradient conditions, respectively.

EXAMPLE 12

[0112]FIG. 14 (panels a, b, c, d, and e) are representativeelectrochromatograms showing plots of absorbance versus retention timeusing an embodiment of the present invention. The plug lengths were 0.23mm (panel a), 7.6 cm (panel b), 22.8 cm (panel c), 45.6 cm (panel d),and 91.2 cm (panel e). The separation solution was 5 mM ammonium acetatein 60% acetonitrile. The sample solutions are: 5 mM ammonium acetate in60% acetonitrile (panel a) and 40% acetonitrile (panels b, c, d, and e).The sample concentrations were 9 to 50 μg/ml (panel a), 0.9 to 5 μg/ml(panels b, c, d, and e). The applied voltage was 22 kV, and absorbancewas detected at 254 nm. The elution order was thiourea (peak 1),decanophenone (peak 2), and pyrene (peak 3).

[0113] The peak heights of decanophenone (peak 2) and pyrene (peak 3)increased with increasing plug lengths. The injection was increased from0.23 mm (panel a) to 7.6 cm (panel b), 22.8 cm (panel c), 45.6 cm (paneld), and 91.2 cm (panel e), which corresponds to 0.1%, 30%, 89%, 178%,and 356% of the total capillary length. The high porosity or the lowresistance to flow of the porous matrix made it possible to introduceincreasing lengths of the sample solution in a rather effortless manner.Longer than 91.2 cm injection is still possible. It is not performed,however, owing to loss of resolution as observed in FIG. 14 (panel e).The electrochromatogram in panel d or e is believed to be the firstdemonstration in CEC showing sample injections longer than the totalcapillary length. The volume of sample injected was also greater than 1μl. A comparison of the peak heights obtained in panels a and e suggestsimprovements in peak heights of 1118 times and 1104 times fordecanophenone and pyrene, respectively. These values are the highestreported sensitivity improvements for neutral analytes using a simpleon-line preconcentration technique in CEC. The strong interaction of theanalytes to the porous matrix and the inherent rapid mass transfercharacteristics of the porous matrix allowed for the observation of suchmarked preconcentration effects.

[0114] Successful separations have been done with PSG in 250-μm i.d.capillaries (data not shown). This work opens the possibility ofperforming semi-preparative separations involving long plug injections.Injection volumes in the μl range could easily be made.

EXAMPLE 13

[0115]FIG. 15 (panels a, b, c, d, e, and f) are representativeelectrochromatograms showing plots of absorbance versus retention timeusing an embodiment of the present invention. The plug lengths were 0.1mm (panel a) and 1.8 cm (panels b, c, d, e, and f). Injections were doneusing pressure wherein the injection length is fixed at 1.8 cm. Thesample solutions were the same as the separation solution (panels a andb), 10 mM phosphoric acid in 20% acetonitrile (panel c), 10 mMphosphoric acid in 70% acetonitrile (panel d), 50 mM phosphoric acid in40% acetonitrile (panel e), 0.05 mM phosphoric acid in 40% acetonitrile(panel f). The separation solution was 10 mM phosphoric acid in 40%acetonitrile. The peptide concentrations were 16.7 μg/ml each, theapplied voltage was 12 kV, and the absorption detection was at 214 nm.The elution order was bradykinin (peak 1), angiotensin II (peak 2),tripeptide I (peak 3), tripeptide II (peak 4), and methionine enkephalin(peak 5).

[0116] Although it was expected that the peak shapes would be betterunder a gradient condition (FIG. 15, panel c) as compared to anongradient one (FIG. 15, panel b), the resulting peak shapes werebetter using a higher concentration of acetonitrile in the sample matrix(FIG. 15, panel d). Better peak shapes were observed in FIG. 15 (paneld) resulting from sample stacking. The broadening effect of using ahigher concentration of the eluting solvent in the sample solution(reverse gradient effect due to higher concentration of eluting solvent)is not observed because the cationic peptides immediately migrate to theseparation buffer once voltage is applied, thus the peptide zones arealready in the separation solution before it reaches the porous matrix.Sample stacking is also shown in FIG. 15 (panel f) where the sample isprepared in a matrix having a lower concentration of buffer componentand a similar percentage of acetonitrile compared to the separationsolution.

[0117] Undesirable peak shapes are observed in FIG. 15 (panel c)resulting from destacking. Destacking is the broadening of chargedanalytes when analytes pass the concentration boundary that separatesregions of low and high electric field strengths. The low electric fieldzone is the high conductivity sample matrix containing more water.Destacking is also shown in FIG. 15 (panel e) where the sample isprepared in a matrix having a higher concentration of buffer componentand a similar percentage of acetonitrile compared to the separationsolution.

EXAMPLE 14

[0118]FIG. 16 (panels a and b) are representative electrochromatogramsshowing plots of absorbance versus retention time using an embodiment ofthe present invention. The injections were 0.1 mm using 0.5 psi pressure(panel a) and 15-s at 5 kV (panel b). The sample solutions were 10 mMphosphoric acid in 40% acetonitrile (panel a) and 0.5 μM phosphoric acidin 40% acetonitrile (panel b). The peptide concentrations were 16.7μg/ml each (panel a) and 167 ng/ml each (panel b). The separationvoltage was 12 kV. Other conditions are the same as in Example 13.

[0119] The analytes in FIG. 16 (panel b) were one hundred times lessconcentrated than those in FIG. 16 (panel a). Improvements in peakheights for bradykinin (peak 1), angiotensin II (peak 2), tripeptide I(peak 3), tripeptide II (peak 4), and methionine enkephalin (peak 5)were 1040, 820, 810, 950, and 711 times, respectively. For thepreconcentration procedure, % RSDs (n=5) of peak heights ranged from6.2% to 16.2% while % RSDs (n=5) of migration time ranged from 0.7% to1.5%. Reproducibility of peak heights should be improved with the use ofan internal standard.

[0120] Field enhanced sample injection was performed by dissolving thesample in a low conductivity matrix (0.5 μM phosphoric acid in 40%acetonitrile), followed by injection using voltage with the negativeelectrode at the detector end. As the voltage was applied, the lowconductivity sample matrix enters the capillary by virtue ofelectroosmotic flow (EOF) while the cationic peptides enter the columnby virtue of both EOF and electrophoretic flow. Only a very small plugof sample matrix was introduced because the low pH of the separationsolution markedly decreases the EOF, which prevents the dissociation ofsilanol groups at the capillary walls. An unretained neutral solute(thiourea) was actually detected after 30 minutes.

[0121] The electric field in the sample matrix zone introduced into thecolumn was much higher than the separation zone. This effect caused thehigh electrophoretic velocity of the cationic peptides entering thecapillary. The high analyte electrophoretic velocity caused a largeamount of peptides to be introduced, unlike in hydrodynamic injection,the volume of sample loaded limited the amount of sample introduced. Thehigh analyte electrophoretic velocity also caused focusing orpreconcentration of peptides at the concentration boundary between thesample matrix and separation solution (sample stacking). Introduction ofa water plug before electrokinetic injection, which is suggested to beuseful in sample stacking with electrokinetic injection, did not improvethe peak heights because of the similar direction of the EOF and analyteelectrophoretic velocities. The low conductivity sample matrix thatenters the capillary also maintains the enhancement of the electricfield at the inlet end of the capillary during injection.

[0122] With the conditions in FIG. 16, optimum electrokinetic injectiontime at 5 kV was found to be 15 s. Longer injections lead to broadeningof the peaks. After the injection, the separation voltage was appliedwith the same polarity as in the injection (negative electrode at thedetector side). The analytes moved to the cathode and were subsequentlypreconcentrated again based on their retention on the PSG column. Themethod was considered selective for cations because cations were mostlyintroduced into the capillary. The injected neutrals migrated after theunretained neutral marker and the cations because the EOF was very slow.At the pH used, all the analytes were either positively charged orneutral. Applicability of the technique to other cationic samples isalso possible.

EXAMPLE 15

[0123] Table 2 lists the types and volumes of reagents used to makedifferent precursor stock solutions where the ratio of the acid catalystto the precursor, methacryloxypropyltriethoxysilane, was varied or wherethe precursor was reacted with a co-precursor (to form a mixed phase PSGmonolith). TABLE 2 Volume (μL) Solution Precursor¹ BTE² BTO³ HCl⁴ A 375 0 0 100 K 375 200 0 100 J 575  0 0 100 M 500  0 75  100 P 375 200 0 100

[0124] Either changing the concentration of the precursor in thereaction solution or using a co-precursor for the formation of mixedphases modified the chemical nature of the parent PSG monolith. The PSGmonoliths, PSG-A and PSG-J, prepared from solutions A and J,respectively, differ only in the volume of precursor used in thereaction with J containing a higher volume of the precursor than A. Ahigher volume of the precursor in the reaction should result in a densermonolith in the capillary column. The PSG monolith, PSG-K, was preparedwith the precursor and bis(triethoxysilyl)ethane as a co-precursor. ThePSG monoliths, PSG-M and PSG-P, were prepared with the precursor anddifferent amounts of bis(triethoxysilyl)octane as the co-precursor. Theco-precursors hydrolyze and condense with the precursor to form hybridsols (mixed phases).

[0125] For solutions A and J, the appropriate volume of the precursorwas added to 100 μL of the acid catalyst (0.12 M HCl), and the resultingsolution was stirred for 15 minutes at room temperature (in the dark).For solutions K, M, and P, the appropriate volume of the precursor wasadded to 100 μL of 0.12 M HCl followed by the addition of theappropriate amount of bis(triethoxysilyl)ethane; the resulting solutionwas stirred for 15 minutes at room temperature (in the dark). All ofthese solutions were used within two hours of their preparation.

[0126] A similar procedure was followed in making the toluene/precursorstock solutions with the photoinitiator added. The amount ofphotoinitiator added to the toluene/precursor stock solution was 10 mgphotoinitiator for every 100 μL of the toluene/precursor stock solution.

[0127] The capillary was prepared and conditioned in the same manner aspreviously described. PSG capillary column conditioning is the same asbefore.

[0128] The separation factors of the monoliths for two test mixtures ofalkyl phenyl ketones (APKs) and polycyclic aromatic hydrocarbons (PAHs)were determined. The separation factor is a measure of the analyteseparation capability of a chromatographic system. The separation factoris a is given by k₂/k₁, where k is the retention factor for a particularanalyte, and k₂ and k₁ are the k values for adjacent analytes. Theretention factor k=(t_(R)−t_(o))/t_(o) was determined in the usual way,where t_(R) is the analyte retention time and t_(o) is the retentiontime of an unretained marker, for which we used thiourea. Table 3 liststhe separation factor of each PSG monolith for naphthalene and pyrene.TABLE 3 Monolith k_(n) k_(Py) α_(Npy) R_(s)(N/Py) PSG-A 0.14 0.36 2.572.43 PSG-K 0.23 0.56 2.43 4.09 PSG-J 0.31 0.79 2.55 4.35 PSG-M 0.35 0.902.57 4.37 PSG-P 0.25 0.69 2.76 9.03

[0129] The values for a varied from 2.43 for PSG-K to 2.76 for PSG-Pwith the separation factor for PSG-P being slightly higher than that ofthe other monoliths. Separation factors greater than 1 indicatedsuccessful separation of the analytes.

[0130]FIG. 17 (panels a, b, c, d, and e) are representativeelectrochromatograms showing plots of absorbance versus retention timeusing an embodiment of the present invention. A mixture of thiourea (T),naphthalene (N), phenanthrene (Ph), and pyrene (Py) were separated onPSG-A (FIG. 17, panel a), PSG-K (FIG. 17, panel b), PSG-J (FIG. 17,panel c), PSG-M (FIG. 17, panel d), and PSG-P (FIG. 17, panel e). In allcases the peaks of the different analytes were well resolved.

[0131] Resolution was determined from the expression${R_{s} = {\frac{\sqrt{N}}{4}\frac{\left( {\alpha - 1} \right)}{\alpha}\frac{k}{\left( {k + 1} \right)}}},$

[0132] where N is the efficiency (theoretical plate number), a is theseparation factor, and k the retention factor for a particular analyte.PSG-A has the lowest resolution of 2.43 for naphthalene and pyrenewhereas PSG-J has a resolution of 4.35 for the same two analytes. Thehigher volume of the precursor used in the preparation of PSG-J ascompared to PSG-A resulted in increased hydrophobicity of the monolith.The retention factors for naphthalene and pyrene on PSG-J were 0.31 and0.79, respectively, and these values reflected the increase in thehydrophobicity of the monolith. These values represent increases of 55%and 54%, respectively.

[0133] The use of the co-precursor, bis(triethoxysilyl)octane in PSG-Mand bis(triethoxysilyl)ethane in PSG-K and PSG-P resulted in resolutionfor naphthalene and pyrene of 4.37, 4.09, and 9.03, respectively, whichis an enhancement of up to 73% as compared to the resolution on theparent PSG-A (Rs=2.43). The retention factors for naphthalene (0.23) andpyrene (0.56) were both 60% higher for these three monoliths than forPSG-A.

EXAMPLE 16

[0134] The separation column was prepared as described above in Example15 for monolith PSG-J. For a porous matrix having a length of 15 cm, theretention factors for napthalene and pyrene, k_(N) and k_(Py),respectively, were 0.31 and 0.79, respectively, for a porous matrix madewith 80% toluene. For a similar porous matrix having a length of 10 cm,k_(N) and k_(Py) were 0.10 and 0.24, respectively. There was a linearrelationship between length and k_(N) (r=0.991) and k_(Py) (r=0.991).The separation factors for 15-cm, 10-cm, and 5-cm porous matrices were2.55, 2.52, and 2.40, respectively. Thus, for the shortest monolithlength, a high separation factor was maintained, while the elution timesfor the analytes were significantly reduced. Decreasing the length ofthe porous matrix in a capillary column led to a decrease in the elutiontimes of the test analytes. Decreasing this length had an effect ofdecreasing the retention factors of naphthalene and pyrene.

EXAMPLE 17

[0135] The separation column was prepared as described above in Example15. For PSG-A made with 80% toluene, k_(N) was 0.14 and k_(Py) is 0.36,whereas k_(N) was 0.30 and k_(Py) was 0.74 for PSG-A made with 73%toluene. The separation factors for PSG-A made with 80% toluene and 73%toluene were 2.57 (0.1%RSD) and 2.47 (0.1%RSD), respectively. The valueof k increased by 53% and 51% for naphthalene and pyrene when 73%toluene was used in the preparation of the monoliths.

[0136] A similar trend was observed for PSG-J where k_(N) was 0.31 andk_(Py) was 0.79 for a monolith made with 80% toluene. The k_(N) (0.49)and k_(Py) (1.23) values increased by 37% and 36% when the concentrationof toluene was decreased from 80% to 73%. The separation factors ofPSG-J made with 80% toluene and 73% toluene were 2.55 and 2.51,respectively.

[0137] The resolution of naphthalene and pyrene differed significantlywhen comparing PSG monoliths made with 80% and 73% toluene. When thepore size decreased, which was brought about by using lower volumes oftoluene, the PSG surface increased with a resulting increase in theretention and resolution under the same separation solution conditions.Thus, the permeability of the porous matrix affects the retention of theanalytes.

[0138] While the invention has been described above by reference tovarious embodiments, it will be understood that changes andmodifications may be made without departing from the scope of theinvention. The present invention includes all that fits within theliteral and equitable scope of the appended claims. All referencesreferred to above are incorporated herein by reference in theirentireties.

What is claimed is:
 1. A separation column comprising: a separationchannel; and a fritless separation medium in the channel, said mediumcomprising a porous matrix, said porous matrix comprising a metalorganic photopolymer.
 2. The column of claim 1, wherein the separationchannel has a channel wall, and the medium is attached to the channelwall and fills at least a section of the channel.
 3. The column of claim1, wherein the porous matrix is homogeneous and contains nochromatographic particles.
 4. The column of claim 1, wherein a precursorof the photopolymer comprises a metal alkoxide.
 5. The column of claim4, wherein the metal alkoxide comprises a metal, and the metal isselected from the group consisting of aluminum, barium, antimony,calcium, chromium, copper, erbium, germanium, iron, lead, lithium,phosphorus, potassium, silicon, tantalum, tin, titanium, vanadium, zinc,and zirconium.
 6. The column of claim 4, wherein the metal alkoxidecomprises at least one photoactive group.
 7. The column of claim 1,wherein the porous matrix has an affinity for an analyte.
 8. The columnof claim 1, wherein the separation medium comprises a homogeneous phase.9. The column of claim 1, wherein the separation channel is a capillaryseparation channel or a planar structure.
 10. A separation columncomprising: a separation channel; and a fritless separation medium inthe channel, said medium comprising a porous matrix, said porous matrixcomprising a metal organic polymer.
 11. A method of preparing a monolithin a separation column, comprising: providing a separation column;introducing a mixture into the column, the mixture comprising a metalorganic compound; and irradiating the mixture, causing the mixture toform a solid, porous matrix via photoinitiated polymerization, therebyforming a fritless separation medium in the column.
 12. The method ofclaim 11, wherein the porous matrix contains no chromatographicparticles.
 13. The method of claim 11, wherein the mixture comprises atleast one metal organic monomer, at least one porogen, and aphotoinitiator.
 14. The method of claim 13, wherein the porogen isselected controllably to form pores in the matrix.
 15. The method ofclaim 14, further comprising selecting a molar ratio of monomer toporogen to form pores in the matrix.
 16. The method of claim 11, whereinthe irradiating comprises irradiating the mixture with visible orultraviolet light.
 17. The method of claim 11, further comprisingintroducing an organic solvent into the column, the column including thesolid, porous matrix.
 18. The method of claim 11, wherein the providingcomprises providing a capillary or a planar structure.
 19. A method ofseparating a sample of analytes, comprising: providing a separationcolumn comprising a separation channel and a fritless separation mediumin the channel, said medium comprising a porous matrix, said porousmatrix comprising a metal organic photopolymer; introducing a sample ofanalytes carried in a solution through the column, wherein the mediumconcentrates the analytes on the column; and causing a solution to flowthrough the column, thereby separating and eluting the analytes.
 20. Themethod of claim 19, wherein the introducing comprises applying a voltageor a pressure to the column.
 21. The method of claim 19, wherein theintroducing comprises introducing a sample of analytes carried in afirst solution through the column, and the causing comprises causing asecond solution to flow through the column, wherein the first solutionis the same solution as the second solution.
 22. The method of claim 19,wherein the introducing comprises introducing a sample of analytescarried in a first solution through the column, wherein the firstsolution comprises an eluting solvent, and the causing comprises causinga second solution to flow through the column, wherein the secondsolution comprising the eluting solvent, and a concentration of theeluting solvent in the first solution is less than a concentration ofthe eluting solvent in the second solution.
 23. The method of claim 22,wherein the introducing comprises introducing a sample of analyteshaving an injection plug length greater than a length of the column. 24.The method of claim 19, wherein the introducing comprises causing samplestacking.
 25. The method of claim 19, wherein the providing comprisesproviding a separation medium comprising a porous matrix withoutchromatographic particles.
 26. The method of claim 19, wherein theproviding comprises providing a separation column comprising a capillaryor a planar structure.